Diagnosis and treatment of glaucoma

ABSTRACT

Methods of diagnosing glaucoma, and particularly primary congenital glaucoma, by detecting mutations in a gene associated with glaucoma, such as the CYP1B1 gene, are disclosed. Methods include hybridization analysis, such as Southern or Northern analysis, which use hybridization of a mutant nucleic acid probe to the gene associated with glaucoma; direct mutation analysis by restriction digest; sequencing of the gene associated with glaucoma; hybridization of an allele-specific oligonucleotide with amplified genomic DNA; or identification of the presence of mutant proteins encoded by the gene associated with glaucoma. Kits for use in diagnosis of glaucoma are also described. Methods of treatment of glaucoma, including administration of the protein encoded by the gene associated with glaucoma; administration of genes, gene constructs, or other nucleic acid constructs; or administration of other therapeutic agents, are additionally described.

GOVERNMENT FUNDING

This invention was made with Government support under Contract No.EY-11095 awarded by the National Eye Institute and Contract No.MOI-RR-06192 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

RELATED APPLICATION

This application is a Continuation-in-Part application of U.S. Ser. No.08/800,036, filed Feb. 13, 1997, issued as U.S. Pat. No. 5,830,661, theentire teachings of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Glaucoma is a group of ocular disorders, characterized by degenerationof the optic nerve. It is one of the leading causes of blindnessworldwide. One major risk factor for developing glaucoma is familyhistory: several different inherited forms of glaucoma have beendescribed.

Primary congenital or infantile glaucoma (gene symbol: GLC3) is aninherited disorder that accounts for 0.01-0.04% of total blindness. Itis characterized by an improper development of the aqueous outflowsystem of the eye, which leads to elevated intraocular pressure,enlargement of the globe or cornea (i.e., buphthalmos), damage to theoptic nerve, and eventual visual impairment. Pathogenesis of GLC3remains elusive despite efforts to identify a single anatomic defect. Atleast two chromosomal locations associated with the disease have beenidentified: one locus at 2p21 (GLC3A) (Sarfarazi, M. et al., Genomics30:171-177 (1995); and a second locus at 1p36 (GLC3B) (Akarsu, A. N. etal., Hum. Mol. Gen. 5(8):1199-1203 (1996)). Other specific loci,including a region of 6p and chromosome 11, have been excluded (Akarsu,A. N. et al., Am. J. Med. Genet. 61:290-292 (1996)).

Primary open angle glaucoma (gene symbol: GLC1) is a common disordercharacterized by atrophy of the optic nerve resulting in visual fieldloss and eventual blindness. GLC1 has been divided into two majorgroups, based on age of onset and differences in clinical presentation.

Juvenile-onset primary open angle glaucoma (GLC1A) usually manifests inlate childhood or early adulthood. The progression of GLC1A is rapid andsevere with high intraocular pressure, is poorly responsive to medicaltreatment, and is such that it usually requires ocular surgery. GLC1Ahas been mapped to the q21-q31 region of chromosome 1, with geneticheterogeneity (Sheffield, V. C. et al., Hum. Mol. Genet. 4:1837-1844(1995)).

Adult- or late-onset primary open angle glaucoma (GLC1B) is the mostcommon type of glaucoma. It is milder and develops more gradually thanjuvenile-onset primary open angle glaucoma, with variable onset usuallyafter the age of 40. GLC1B is associated with slight to moderateelevation of intraocular pressure, and often responds satisfactorily toregularly monitored medical treatment. However, because the diseaseprogresses gradually and painlessly, it may not be detected until a latestage when irreversible damage to the optic nerve has already occurred.Linkage, haplotype and clinical data have assigned a locus for GLC1B tothe 2cen-q13 region (Stoilova, D. et al., Genomics 36:142-150 (1996)),as well as a new locus 3q21-q22 (Sarfarazi, M. et al., submitted 1996),with further evidence for several other loci.

Because of the insidious nature of glaucoma, a need remains for a betterand earlier means to diagnose or predict the likelihood of developmentof glaucoma, so that preventative or palliative measures can be takenbefore significant damage to the optical nerve occurs.

SUMMARY OF THE INVENTION

The invention pertains to methods of diagnosing or treating glaucoma.The methods of diagnosing glaucoma in an individual include detectingthe presence of a mutation in a gene associated with the disease. Themutation can be the insertion or deletion of one or more nucleotides,resulting in a frame shift mutation; the change of at least onenucleotide, resulting in a change in the encoded amino acid; the changeof at least one nucleotide, resulting in a premature stop codon; theinsertion of one or several nucleotides, such as an insertion caused byunequal recombination or gene conversion, resulting in an interruptionof the coding sequence of the gene; duplication of a part of the gene;transposition of all or a part of the gene; or rearrangement of all or apart of the gene. More than one mutation can be present in a geneassociated with glaucoma. The mutations associated with glaucoma can beidentified by numerous methods, such as Southern analysis of genomicDNA; amplification of genomic DNA followed by direct mutation analysisby restriction enzyme digestion; Northern analysis of RNA; geneisolation and direct sequencing; or analysis of the protein encoded bythe gene associated with glaucoma.

For example, a sample of DNA containing the gene is obtained from anindividual suspected of having glaucoma or of being a carrier forglaucoma (the test individual). The DNA is contacted with at least onemutant nucleic acid probe under conditions sufficient for specifichybridization of the gene to the mutant nucleic acid probe. The mutantnucleic acid probe comprises DNA, cDNA, or RNA of the gene, or afragment of the gene, having at least one of the mutations describedabove, or an RNA fragment corresponding to such a cDNA fragment. Thepresence of specific hybridization of the mutant nucleic acid fragmentto the mutant nucleic acid probe is indicative of a mutation in the genethat is associated with glaucoma. In another example, the DNA iscontacted with a PNA probe under conditions sufficient for specifichybridization of the gene to the PNA probe; the presence of specifichybridization is indicative of a mutation in the gene that is associatedwith glaucoma.

Alternatively, direct mutation analysis by restriction digest of asample of genomic DNA, RNA or cDNA from the test individual can beconducted, if the mutation results in the creation or elimination of arestriction site. The digestion pattern of the relevant DNA, RNA or cDNAfragment indicates the presence or absence of the mutation associatedwith glaucoma.

The presence of a mutation associated with glaucoma can also bediagnosed by sequence data. A sample of genomic DNA, RNA or cDNA fromthe test individual is obtained, and the sequence of the gene, or afragment of the gene, is determined. The sequence of the gene from theindividual is compared with the known sequence of the gene (the controlsequence). The presence of a mutation, as described above, in the geneof the individual is indicative of the a mutation that is associatedwith glaucoma.

The invention additionally pertains to methods of diagnosing glaucoma inan individual by detecting alterations in expression of a proteinencoded by the gene associated with glaucoma. The alteration inexpression can be an alteration of the amount of protein expressed (aquantitative alteration), or an alteration of the composition of theprotein expressed (a qualitative alteration), or both. An alteration inexpression of the protein encoded by the gene associated with glaucomain a test sample, as compared with expression of protein encoded by thegene associated with glaucoma in a control sample, is indicative of thedisease. Alterations in expression of the protein can be assessed usingstandard techniques, such as Western blotting.

The invention additionally pertains to antibodies (monoclonal orpolyclonal) to proteins encoded by mutated genes associated withglaucoma. These antibodies can also be used in methods of diagnosis. Forexample, a test sample which includes the protein of interest iscontacted with antibodies specific for a protein that is encoded by agene having a mutation associated with glaucoma, as described above.Specific binding of the antibody to the protein of interest isindicative of a mutation associated with glaucoma.

The invention also pertains to methods of treating glaucoma, such asadministration of a therapeutic agent that replaces, mimics orsupplements the activity of the protein encoded by the gene associatedwith glaucoma, as well as methods of gene therapy for glaucoma.

The current invention facilitates identification of mutations in thegenes which are associated with glaucoma, and thereby facilitates bothbetter and earlier diagnosis and treatment of the disease.Identification of such mutations distinguishes one form of glaucoma fromother forms, thereby enabling better treatment planning for affectedindividuals, as well as for other family members who may be affectedindividuals or disease carriers.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a depiction of the GLC3A critical candidate chromosomal regionfor genes associated with primary congenital glaucoma.

FIG. 2 is a depiction of the genomic structure of the CYP1B1 gene, withthree mutations associated with glaucoma identified.

FIG. 3 is a series of diagrams, individually labeled as FIG. 3A, FIG.3B, and FIG. 3C, depicting the analysis of genomic mutations in fiveGLC3A families. FIG. 3A depicts a 13 base pair deletion in families 17and 26 as assayed by acrylamide gel electrophoresis and sequencing; FIG.3B depicts analysis of the single cytosine base insertion in families 10and 11; FIG. 3C depicts PCR characterization of a large deletionobserved in family 15.

DETAILED DESCRIPTION OF THE INVENTION

The current invention relates to methods of diagnosing glaucoma. Theterm "glaucoma", as used herein, refers to inherited glaucomas, such asprimary congenital or infantile glaucoma; primary open angle glaucoma(POAG), including both juvenile-onset and adult- or late-onset POAG;secondary glaucomas; pigmentary glaucoma; and low tension glaucoma.

As described herein, Applicant has identified a gene associated withglaucoma: mutations in the gene are associated with the presence ofdisease. To identify the gene associated with glaucoma, Applicantinvestigated the genes that were present at a locus, GLC3A, that waslinked with primary congenital glaucoma. After identifying candidategenes, Applicant performed direct sequencing analysis of the candidategenes from samples of genomic DNA from individuals in panels of familieswith primary congenital glaucoma. Applicant identified seventeendifferent mutations in the gene for human cytochrome P4501B1 (CYP1B1),as being associated with primary congenital glaucoma. One mutation,found in more than one family, was a 13 base pair deletion that removednucleotides 1410 to 1422 (GAGTGCAGGCAGA (SEQ ID NO. 1)) from the codingsequence of the CYP1B1 gene. This mutation resulted in a frame shiftthat truncated the open reading frame by creating a premature stop codon203 base pairs downstream of the deletions (68 amino acids after thelast original (pre-frame shift) amino acid Thr-354). A second mutationin the CYP1B1 gene that was associated with primary congenital glaucomawas an insertion of an extra cytosine base in a stretch of six cytosineslocated between nucleotides 1209 to 1214. This insertion also resultedin a frame shift mutation that created a premature stop codon 106 basepairs downstream from the site of insertion (36 amino acids downstreamfrom the last original amino acid Pro-289). A third type of mutation inthe CYP1B1 gene also has a deletion that removes part of intron II andmost of the coding sequence of exon III. A fourth mutation was a tenbase pair duplication of nucleotides 1546-1555 (TCATGCCACC, SEQ ID NO.20); this duplication of ten base pairs resulted in a frame shiftmutation that created a premature stop after amino acid 403, removing140 amino acids from the full-length (non-mutant) protein. A fifthmutation was a single base deletion of a cytosine at nucleotide 1737,resulting in a frame shift that created a premature stop codon TAG,resulting in the removal of 80 amino acids (a deletion of all aminoacids after amino acid 463). A sixth mutation was a single base change(a G→T transition) at nucleotide 1187, which resulted in a premature TAAstop codon. A seventh mutation was a change in single base change (a C→Ttransition) at nucleotide 1482, which resulted in a change of theencoded amino acid from proline to leucine. An eighth mutation was asingle base change (a G→C transition) at nucleotide 517, which resultedin a change of the encoded amino acid from tryptophan to cysteine. Aninth mutation was a single base change (a G→A transition) at nucleotide528, which resulted in a change of the encoded amino acid from glycineto glutamic acid. A tenth mutation was an insertion of a thymine base atnucleotide 846, causing in a frame shift mutation that resulted in thedeletion of 377 amino acids from the carboxy terminus of the protein. Aneleventh mutation was a single base change (a G→T transition) atnucleotide 1439, which resulted in a change of the encoded amino acidfrom glycine to tryptophan. A twelveth mutation was a single base change(a G→A transition) at nucleotide 1505, resulting in a change of theencoded amino acid from glutamic acid to lysine. A thirteenth mutationwas a single base change (a G→A transition) at nucleotide 1515,resulting in a change of the encoded amino acid from arginine tohistidine. A fourteenth mutation was a single base change (a C→Ttransition) at nucleotide 1656, which resulted in a change of theencoded amino acid from proline to leucine. A fifteenth mutation was adeletion of a single base (G) at nucleotide 1691, causing a frame shiftmutation that resulted in the deletion of 95 amino acids from thecarboxy terminus of the protein. A sixteenth mutation was a single basechange (a C→T transition) at nucleotide 1751, resulting in a change ofthe encoded amino acid from arginine to tryptophan. A seventeenthmutation was a duplication of 27 nucleotides beginning at nucleotide1749 (i.e., duplication of nucleotides 1749-1775), causing a frame shiftmutation that resulted in deletion of 76 amino acids from the carboxyterminus of the protein.

As a result of the discovery of these mutations in a gene associatedwith glaucoma, methods of diagnosing glaucoma are now available. Usingmethods such as those described herein, or other appropriate methods, itis now possible to identify genes associated with glaucoma. A "geneassociated with glaucoma" is a gene that, if mutated, has a mutationthat is associated with glaucoma. The "gene associated with glaucoma"includes the DNA encoding a protein, as well as other components, suchas leader and trailer sequences, promoter elements, introns and exons."Mutations associated with glaucoma", as described herein, includemutations in the gene as well as mutations in the cDNA or mRNA of thegene, wherein the mutations in the gene (or the cDNA or mRNA of thegene) have been determined to be associated with glaucoma, such as bylinkage analysis (or by direct sequencing).

To identify other genes associated with glaucoma, analysis of loci knownto be associated with glaucoma can be performed: genes within or closelylinked to the locus of interest (the locus identified as beingassociated with glaucoma) can be analyzed for mutations, using methodssuch as those described below. For example, several loci have beenassigned to adult-onset primary open angle glaucoma (POAG), includingthe 2cen-q13 region (Stoilova, D. et al., Genomics 26:142-150 (1996));the 3q21-q22 region (Sarfarazi, M. et al., submitted 1996); 8q24(Trifan, et al., in preparation); and 10p (Sarfarazi, M. et al., inpreparation). These loci can be investigated to identify genesassociated with glaucoma. Alternatively, new loci can be identified bygenetic analysis of kindreds affected by glaucoma; these loci can thenbe analyzed to identify genes associated with glaucoma. These methodsare applicable to all forms of glaucoma for which there is a geneticbasis.

After identification of a gene associated with glaucoma, diagnosis ispossible. Diagnosis of glaucoma is made by detecting a mutation ormutations in a gene associated with glaucoma. The mutation in the geneassociated with glaucoma can be the insertion or deletion of a singlenucleotide, or of more than one nucleotide, resulting in a frame shiftmutation; the change of at least one nucleotide, resulting in a changein the encoded amino acid; the change of at least one nucleotide,resulting in the generation of a premature stop codon; the deletion ofseveral nucleotides, resulting in a deletion of one or more amino acidsencoded by the nucleotides; the insertion of one or several nucleotides,such as by unequal recombination or gene conversion, resulting in aninterruption of the coding sequence of the gene; duplication of all or apart of the gene; transposition of all or a part of the gene; orrearrangement of all or a part of the gene. More than one such mutationmay be present in a single gene. Such sequence changes cause a mutationin the protein encoded by the gene associated with glaucoma. Forexample, if the mutation is a frame shift mutation, the frame shift canresult in a change in the encoded amino acids, and/or can result in thegeneration of a premature stop codon, causing generation of a truncatedprotein. Alternatively, a mutation associated with glaucoma can be asynonymous mutation in one or more nucleotides (i.e., a mutation thatdoes not result in a change in the protein encoded by the geneassociated with glaucoma). Such a mutation may alter splicing sites, orotherwise affect the transcription or translation of the gene. A geneassociated with glaucoma that has any of the mutations described aboveis referred to herein as a "mutant gene."

In a first method of diagnosing glaucoma, hybridization methods, such asSouthern analysis, are used (see Current Protocols in Molecular Bioloay,Ausubel, F. et al., eds., John Wiley & Sons, including all supplementsthrough 1997). For example, a test sample of genomic DNA, RNA, or cDNA,is obtained from an individual suspected of having (or carrying a defectfor) glaucoma (the "test individual"). The individual can be an adult,child, or fetus. The test sample can be from any source which containsgenomic DNA, such as a blood or tissue sample, such as from skin orother organs. In a preferred embodiment, the test sample of DNA isobtained from a fibroblast skin sample, from hair roots, or from cellsobtained from the oral cavity (e.g., via mouthwash). In anotherpreferred embodiment, the test sample of DNA is obtained from fetalcells or tissue by appropriate methods, such as by amniocentesis orchorionic villus sampling. The DNA, RNA, or cDNA sample is examined todetermine whether a mutation associated with glaucoma is present; thepresence of the mutation is indicated by hybridization of the gene inthe genomic DNA, RNA, or cDNA to a nucleic acid probe. A "nucleic acidprobe", as used herein, can be a DNA probe or an RNA probe. The nucleicacid probe hybridizes to at least one of the mutations associated withglaucoma, as described above. A fragment of such a nucleic acid probecan also be used, provided that the fragment hybridizes to the part ofthe gene that contains the mutation.

To diagnose glaucoma by hybridization, a hybridization sample is formedby contacting the test sample containing a gene associated withglaucoma, with at least one nucleic acid probe. The hybridization sampleis maintained under conditions which are sufficient to allow specifichybridization of the nucleic acid probe to the gene associated withglaucoma. "Specific hybridization", as used herein, indicates exacthybridization (e.g., with no mismatches). Specific hybridization can beperformed under high stringency conditions or moderate stringencyconditions, for example. "Stringency conditions" for hybridization is aterm of art which refers to the conditions of temperature and bufferconcentration which permit hybridization of a particular nucleic acid toanother nucleic acid in which the first nucleic acid may be perfectlycomplementary to the second, or the first and second nucleic acids mayshare only some degree of complementarity. For example, certain highstringency conditions can be used which distinguish perfectlycomplementary nucleic acids from those of less complementarity. "Highstringency conditions" and "moderate stringency conditions" for nucleicacid hybridizations are explained in chapter 2.10 and 6.3, particularlyon pages 2.10.1-2.10.16 and pages 6.3.1-6 in Current Protocols inMolecular Biology, supra, the teachings of which are hereby incorporatedby reference. The exact conditions which determine the stringency ofhybridization depend on factors such as length of nucleic acids, basecomposition, percent and distribution of mismatch between thehybridizing sequences, temperature, ionic strength, concentration ofdestabilizing agents, and other factors. Thus, high or moderatestringency conditions can be determined empirically. In one embodiment,the hybridization conditions for specific hybridization are moderatestringency. In a particularly preferred embodiment, the hybridizationconditions for specific hybridization are high stringency.

Specific hybridization, if present, is then detected using standardmethods. If specific hybridization occurs between the nucleic acid probeand the gene associated with glaucoma in the test sample, then the geneassociated with glaucoma has a mutation. More than one nucleic acidprobe can also be used concurrently in this method. Specifichybridization of any one of the nucleic acid probes is indicative of amutation in the gene that is associated with glaucoma, and is thereforediagnostic for the disease.

For example, in the diagnosis of primary congenital glaucoma, a nucleicacid probe can be prepared that hybridizes to a part of the CYP1B1 genehaving a 13 base pair deletion of nucleotides 1410 to 1422. If thisnucleic acid probe specifically hybridizes with the gene associated withglaucoma in the test sample, a diagnosis of primary congenital glaucomais made. Alternatively, a nucleic acid probe can be prepared thathybridizes to a CYP1B1 gene having one of the other mutations describedabove, such as: an extra cytosine (C) base located in the stretch of sixcytosines between nucleotides 1209 and 1214; a 10 base pair duplicationof nucleotides 1546-1555; a deletion of cytosine (C) at nucleotide 1737;a G→T transition at nucleotide 1187; a C→T transition at nucleotide1482; a G→C transition at nucleotide 517; a G→A transition at nucleotide528; an insertion of thymine (T) at nucleotide 846; a G→T transition atnucleotide 1439; a G→A transition at nucleotide 1505; a G→A transitionat nucleotide 1515; a C→T transition at nucleotide 1656; a deletion ofguanine (G) at nucleotide 1691; a C→T transition at nucleotide 1751; ora duplication of 27 nucleotides beginning with nucleotide 1749. Specifichybridization of such a nucleic acid probe with the gene associated withglaucoma in the test sample is indicative of primary congenitalglaucoma.

In another hybridization method, Northern analysis (see CurrentProtocols in Molecular Biology, Ausubel, F. et al., eds., John Wiley &Sons, supra) is used to identify the presence of a mutation associatedwith glaucoma. For Northern analysis, a sample of RNA is obtained fromthe test individual by appropriate means. Specific hybridization of anucleic acid probe, as described above, to RNA from the individual isindicative of a mutation in the gene that is associated with glaucoma,and is therefore diagnostic for the disease.

For representative examples of use of nucleic acid probes, see, forexample, U.S. Pat. Nos. 5,288,611 and 4,851,330.

Alternatively, a peptide nucleic acid (PNA) probe can be used instead ofa nucleic acid probe in the hybridization methods described above. PNAis a DNA mimic having a peptide-like, inorganic backbone, such asN-(2-aminoethyl)glycine units, with an organic base (A, G, C, T or U)attached to the glycine nitrogen via a methylene carbonyl linker (see,for example, Nielsen, P. E. et al., Bioconjugate Chemistry, 1994, 5,American Chemical Society, p. 1 (1994). The PNA probe can be designed tospecifically hybridize to a gene having a mutation associated withglaucoma. Hybridization of the PNA probe to the mutant gene associatedwith glaucoma is diagnostic for the disease.

In another method of the invention, mutation analysis by restrictiondigestion can be used to detect mutant genes, if the mutation in thegene results in the creation or elimination of a restriction site. Atest sample containing genomic DNA is obtained from the test individual.Polymerase chain reaction (PCR) can be used to amplify the geneassociated with glaucoma (and, if necessary, the flanking sequences) ina test sample of genomic DNA from the test individual. RFLP analysis isconducted as described (see Current Protocols in Molecular Biology,supra). The digestion pattern of the relevant DNA fragment indicates thepresence or absence of the mutation associated with glaucoma.

Sequence analysis can also be used to detect specific mutations in thegene. A test sample of DNA is obtained from the test individual. PCR canbe used to amplify the gene, and/or its flanking sequences. The sequenceof the gene associated with glaucoma, or a fragment of the gene, isdetermined, using standard methods. The sequence of the gene (or genefragment) is compared with the known nucleic acid sequence of the gene.The presence of any of the mutations associated with glaucoma, asdescribed above, indicates that the individual is affected with, or is acarrier for, glaucoma. In one embodiment of this method, such sequenceanalysis can be used to identify mutations in the CYP1B1 gene that areassociated with glaucoma.

Allele-specific oligonucleotides can also be used to detect the presenceof a mutation associated with glaucoma, through the use of dot-blothybridization of amplified gene products with allele-specificoligonucleotide (ASO) probes (see, for example, Saiki, R. et al.,(1986), Nature (London) 324:163-166). An "allele-specificoligonucleotide" (also referred to herein as an "allele-specificoligonucleotide probe") is an oligonucleotide of approximately 10-50base pairs, preferably approximately 15-30 base pairs, that specificallyhybridizes to the gene that contains a mutation associated withglaucoma. An allele-specific oligonucleotide probe that is specific forparticular mutations in the gene associated with glaucoma can beprepared, using standard methods (see Current Protocols in MolecularBioloay, supra). To identify mutations in the gene that are associatedwith glaucoma, a test sample of DNA is obtained from the testindividual. PCR can be used to amplify all or a fragment of the geneassociated with glaucoma, and its flanking sequences. The DNA containingthe amplified gene associated with glaucoma (or fragment of the gene) isdot-blotted, using standard methods (see Current Protocols in MolecularBiology, supra), and the blot is contacted with the oligonucleotideprobe. The presence of specific hybridization of the probe to theamplified gene associated with glaucoma is then detected. Specifichybridization of an allele-specific oligonucleotide probe to DNA fromthe individual is indicative of a mutation in the gene associated withglaucoma that is associated with glaucoma, and is therefore diagnosticfor the disease.

Diagnosis of glaucoma can also be made by examining expression of theprotein encoded by the gene associated with glaucoma. A test sample froman individual is assessed for the presence of an alteration in theexpression of the gene associated with glaucoma. An alteration inexpression of a protein encoded by a gene associated with glaucoma canbe an alteration in the quantitative protein expression (i.e., theamount of protein produced); an alteration in the qualitative proteinexpression (i.e., the composition of the protein), or both. An"alteration" in the protein expression, as used herein, refers to analteration is a test sample as compared with the expression of proteinby a gene associated with glaucoma in a control sample. A control sampleis a sample that corresponds to the test sample (e.g., is from the sametype of cells), and is from an individual who is not affected byglaucoma. An alteration in the expression of the protein in the testsample, as compared with the control sample, is indicative of glaucoma.Various means of examining expression of protein encoded by the geneassociated with glaucoma can be used, including spectroscopy,colorimetry, electrophoresis, isoelectric focusing, and immunoblotting(see Current Protocols in Molecular Biology, particularly chapter 10).For example, Western blotting analysis, using an antibody thatspecifically binds to a protein encoded by a mutant gene, or an antibodythat specifically binds to a protein encoded by a non-mutant gene, canbe used to identify the presence in a test sample of a protein encodedby a mutant gene associated with glaucoma, or the absence in a testsample of a protein encoded by a non-mutant gene. The presence of aprotein encoded by a mutant gene, or the absence of a protein encoded bya non-mutant gene, is diagnostic for glaucoma.

In one embodiment of this method, the level or amount of protein encodedby a gene associated with glaucoma in a test sample is compared with thelevel or amount of the protein encoded by the gene associated withglaucoma in a control sample. A level or amount of the protein in thetest sample that is higher or lower than the level or amount of theprotein in the control sample, such that the difference is statisticallysignificant, is indicative of an alteration in the expression of theprotein encoded by the gene associated with glaucoma, that is associatedwith disease. Alternatively, the composition of the protein encoded by agene associated with glaucoma in a test sample is compared with thecomposition of the protein encoded by the gene associated with glaucomain a control sample. A difference in the composition of the protein inthe test sample, as compared with the composition of the protein in thecontrol sample, is indicative of glaucoma. In another embodiment, boththe level or amount and the composition of the protein can be assessedin the test sample and in the control sample. A difference in the amountor level of the protein in the test sample, compared to the controlsample; a difference in composition in the test sample, compared to thecontrol sample; or both a difference in the amount or level, and adifference in the composition, is indicative of disease.

The invention also relates to antibodies to mutant proteins encoded bygenes associated with glaucoma. A "mutant protein", as referred toherein, is a protein or protein fragment that is encoded by a mutantgene associated with glaucoma. Once a mutation in a gene associated withglaucoma has been identified, the protein or protein fragment encoded bythe mutated gene (also referred to herein as the protein of interest)can be identified, and antibodies can be raised to the protein orprotein fragment using standard methods (see, for example, CurrentProtocols in Molecular Biology, supra). The term "antibody", as usedherein, encompasses both polyclonal and monoclonal antibodies, as wellas mixtures of more than one antibody reactive with the protein orprotein fragment (e.g., a cocktail of different types of monoclonalantibodies reactive with the mutant protein or protein fragment). Theterm antibody is further intended to encompass whole antibodies and/orbiologically functional fragments thereof, chimeric antibodiescomprising portions from more than one species, humanized antibodies,human-like antibodies, and bifunctional antibodies. Biologicallyfunctional antibody fragments are those fragments sufficient for bindingof the antibody fragment to the protein of interest.

Monoclonal antibodies (mAb) reactive with a mutant protein encoded by agene associated with glaucoma can be produced using somatic cellhybridization techniques (Kohler and Milstein, Nature 256:495-497(1975)) or other techniques. In a typical hybridization procedure, acrude or purified mutant protein encoded by a gene associated withglaucoma can be used as the immunogen. An animal is immunized with theimmunogen to obtain antibody-producing spleen cells. The species ofanimal immunized will vary depending on the specificity of mAb desired.The antibody producing cell is fused with an immortalizing cell (e.g., amyeloma cell) to create a hybridoma capable of secreting antibodies tothe mutant protein of the invention. The unfused residualantibody-producing cells and immortalizing cells are eliminated.Hybridomas producing desired antibodies are selected using conventionaltechniques and the selected hybridomas are cloned and cultured.

Polyclonal antibodies can be prepared by immunizing an animal in asimilar fashion as described above for the production of monoclonalantibodies. The animal is maintained under conditions whereby antibodiesare produced that are reactive with the mutant protein encoded by thegene associated with glaucoma. Blood is collected from the animal uponreaching a desired titer of antibodies. The serum containing thepolyclonal antibodies (antisera) is separated from the other bloodcomponents. The polyclonal antibody-containing serum can optionally befurther separated into fractions of particular types of antibodies(e.g., IgG, IgM).

Antibodies that specifically bind to a protein or protein fragmentencoded by the mutant gene associated with glaucoma (i.e., those thatbind to the protein or protein fragment encoded by the mutant gene, butnot to protein encoded by a non-mutant copy of the gene) can also beused in methods of diagnosis. A test sample containing the proteinencoded by the gene associated with glaucoma is contacted with theantibody; binding of the antibody to the protein is indicative of thepresence of a protein encoded by the mutant gene, and is diagnostic fordisease.

The present invention also includes kits useful in the methods of theinvention. The kits can include a means for obtaining a test sample;nucleic acid probes, PNA probes, or allele-specific oligonucleotideprobes; appropriate reagents; antibodies to mutant proteins encoded bygenes associated with glaucoma; instructions for performing the methodsof the invention; control samples; and/or other components.

The invention also pertains to modes of therapy to treat glaucoma.Glaucoma can be treated by administration of an agent that, whenadministered, ameliorates, relieves, lessens the severity of, oreliminates the symptoms of glaucoma. For example, an antibody thatspecifically binds to the mutant protein encoded by the gene associatedwith glaucoma can be administered, in order to reduce or eliminateactivity by the mutant protein. Alternatively or in addition, thenon-mutant protein encoded by the gene associated with glaucoma can beadministered as a therapeutic agent to treat glaucoma. In anotherembodiment, an agent that mimics the activity of the protein encoded bythe gene associated with glaucoma (the mutant protein) can beadministered, in order to supplement or supplant the activity of aprotein encoded by the mutant gene associated with glaucoma. Forexample, peptides which have the same biological activity as the proteinencoded by the gene associated with glaucoma can be used.Peptidomimetics (molecules which are not polypeptides, but which mimicaspects of their structures) can be also designed based on the structureof the protein encoded by the gene associated with glaucoma.Polysaccharides can be prepared that have the same functional groups asthe protein, and which have the same function as the protein.Alternatively, libraries of agents, such as those that can beconstructed using well-known methods of combinatorial chemistry, can beassayed for additional agents. Such agents can be isolated by standardmethods, such as by interaction of the agent with an antibody that alsospecifically binds to the protein encoded by the gene associated withglaucoma.

In another embodiment, an agent that induces or enhances expression of arelated gene, such that expression of the protein encoded by the relatedgene supplements or supplants activity of the mutant protein, can beused. Replacement or supplementation of the activity of the mutantprotein will reduce or eliminate the physiological cause of glaucoma,and thereby treat the disease. Such agents can include proteins,peptides, peptidomimetics, antibodies, or other small molecules thatinduce or enhance expression of a related gene. For example, an agentthat induces or enhances expression of another protein in the cytochromep450 family can be used to treat congenital glaucoma, by supplementingor replacing the activity of a mutant CYP1B1 gene. Alternatively, an DNAconstruct that induces or enhances expression of a related gene can begenerated, such as by the methods described in WO 95/31560, for example.

Alternatively or in addition, an antibody that specifically binds to themutant protein encoded by the gene associated with glaucoma can beadministered before, after, or concurrently with any of the agentsdescribed above, in order to target the mutant protein and reduce oreliminate its activity.

Glaucoma can also be treated by the administration of genes, genetransfer vectors, or other nucleic acid constructs. A non-mutant copy ofthe gene (or cDNA of the gene) associated with glaucoma, or mRNA of anon-mutant copy of the gene (or cDNA) associated with glaucoma, can beprovided to the individual. For example, a gene transfer vectorcontaining a non-mutant gene associated with glaucoma can beadministered, to express the non-mutant gene in the individual affectedby glaucoma. The gene transfer vector can also contain tissue-specificpromoters, as well as other elements (e.g., enhancer elements, splicingsignals, termination and polyadenylation signals, viral replicons,bacterial plasmid sequences, or other vector nucleic acid sequences).Delivery of the vector can be targeted to particular regions or celltypes (e.g., by the use of decorated liposomes, or by introducing thevector in a specific region of the body). Alternatively, purified DNA ormRNA can be used as a therapeutic agent, as described in WO 93/19183 orin WO 90/11092. These methods can also be used to introduce a relatedgene, such that expression of the protein encoded by the related genesupplements or supplants activity of the mutant protein.

In another embodiment, a nucleic acid construct that targets the mutantgene associated with glaucoma, and "corrects" the mutation byintegration or by homologous recombination, can be used. Constructs thatuse homologous recombination to provide DNA encoding a therapeuticprotein or peptide are described in WO 93/09222, for example. Genetherapy as described above can target cells in vivo, by administrationof the therapeutic agent directly to the individual. Alternatively, thegene therapy can target cells in vitro, such as cells that have beenremoved from the individual; the treated cells can then be reimplantedinto the individual. The entire teachings of the publications cited inthe above paragraph are incorporated herein by reference.

Therapeutic agents, including the agents described above as well as thegene transfer vectors, DNA, and/or mRNA described above in relation togene therapy, can be administered opthamologically, subcutaneously,intravenously, intramuscularly, topically, orally, rectally, vaginally,nasally, buccally, by inhalation spray, or via an implanted reservoir.In a preferred embodiment, the therapeutic agent is administered to theeye, such as by topical administration (e.g., eye drops or emulsion).They can be administered in dosage formulations containing conventionalnon-toxic pharmaceutically-acceptable carriers, adjuvants and/orvehicles. The form in which the agents are administered (e.g., capsule,tablet, solution, emulsion) will depend at least in part on the route bywhich they are administered. A therapeutically effective amount of theagent is that amount necessary to significantly reduce or eliminatesymptoms associated with glaucoma. The therapeutically effective amountwill be determined on an individual basis and will be based, at least inpart, on consideration of the agent, the individual's size and gender,the severity of symptoms to be treated, the result sought. Thus, thetherapeutically effective amount can be determined by one or ordinaryskill in the art, employing such factors and routine experimentation.

The therapeutically effective amount can be administered in a series ofdoses separated by appropriate intervals, such as hours, days or weeks.Alternatively, the therapeutically effective amount can be administeredin a single dose. The term, "single dose," as used herein, can be asolitary dose, and can also be a sustained release dose, such as by acontrolled-release dosage formulation of a continuous infusion. Otherdrugs can also be administered in conjunction with the agent.

The invention is further illustrated by the following Example.

EXAMPLE Identification of a Gene Associated with Glaucoma ScreeningPanel and Identification of Loci

A screening panel of 17 families with primary congenital glaucoma wasused (Sarfarazi, M. et al., Genomics 30:171 (1995); Turacli, M. E. etal., Int. Ophthamol. 16:359 (1992)). After excluding many genes andcandidate chromosomal regions (Akarsu, A. N. et al., Am. J. Med. Genet61:290 (1996); Akarsu, A. N. et al., Am. J. Med. Genet. 62:102 (1996)),a random screening of the genome assigned two loci, GLC3A (2p21,Sarfarazi, M. et al., Genomics 30:171 (1995)) and GLC3B (1p36, Akarsu,A. N. et al., Hum. Mol. Genetics 5:1199 (1996)) for primary congenitalglaucoma, with evidence for at least one additional unmapped locus forthe condition. The GLC3A locus on 2p21 has recently been confirmed inanother panel of 25 families from Saudi Arabia (Bejjani, B. A. et al.,Am. J. Human Genet. 59 suppl., A212-1216 (1996)). Two additionalfamilies are also identified. therefore, the GLC3A locus emerges as amajor location for this condition, with nearly 85% of the testedfamilies being linked to this site.

Critical recombination events and inspection of the smallest conservedsegment of homozygosity in the affected members of consanguineousfamilies (Sarfarazi, M. et al., Genomics 30:171 (1995)) reduced theGLC3A critical candidate region to approximately 2.5 cM that is flankedby markers D2S2186 and D2S1346. This candidate region is shown inFIG. 1. The directions toward the centromere and telomere are indicated.Loci anchored on the Chromosome 2 Radiation Hybrid map (Hudson, T. etal., Science 270:1945 (1995)) are boxed. The distances (in cR or cM)from top of Chromosome 2 are indicated. The area shaded in grayrepresents the GLC3A candidate region as defined by recombination eventswith markers D2S2186 (Bejjani, B. A. et al., Am. J. Hum. Genet. 59suppl:A212/1216 (1996)) and D2S1356 (Sarfarazi, M. et al., Genomics30:171 (1995)). The dark edged box identifies the smallest segment ofhomozygosity observed in our consanguineous families. Black horizontalarrows represent the interval within which a particular gene is mapped.

Three genes have been previously mapped to the 2p21 region:non-erythrocytic form of beta-spectrin or beta-fodrin (SPTBN1) (Chang,J. G. et al., Genomics 17:287 (1993); Hu, R. J. et al., J. Biol. Chem.267:18715 (1992)); a guanine nucleotide exchange factor for Ras (hSOS1)(Chardin, P. et al., Science 260:1338 (1993)); Webb, G. C. et al.,Genomics 18:14 (1993)); and interferon-inducible dsRNA-dependent proteinkinase (PRKR) (Barber, G. N. et al., Genomics 16:765 (1993); Squire, J.et al., ibid 16:768 (1993); Hanash, S. M. et al., Genes Chromosom Cancer8:34 (1993)). These genes were implicated as possible candidate genesfor this conditions. The positions of the genes were refined byscreening them against the GeneBridge 4 Radiation Hybrid (RH) panel andmapping them relative to the Whitehead Rh framework (Hudson, T. et al.,Science 270:1945 (1995)). The original MIT order of 91 cell lines wasused. Statistical analysis of the RH data was carried out on the mappingserver at Whitehead Institute for Genome Research. Detailed RH mapinformation can be obtained from http://www-genome.wi.mit.edu. Screeningof the RH panel was carried out by gene specific polymerase chainreaction (PCR) assays. Preferably, the intronic or 3-prime untranslatedsequences were assayed to prevent cross-amplification of the hamster DNAbackground. Map positions for these genes were established as follows(see FIG. 1): SPTBN1 mapped 1.51 centiRay (cR) from marker WI-4077(LOD >3.0); hSOS1 positioned 1.51 cR from marker WI-10326 (LOD >3.0);and PRKR was placed 2.3 cR from D2S177 (LOD >3). therefore, SPTBN1 mapscentromerically to D2S1356 and thus was excluded as a candidate gene forGLC3A.

Inspection of the contig map (Hudson, T. et al., Science 270:1945(1995); Shuler, G. D. et al, Science 274:540 (1996)) harboring the GLC3Alocus revealed that marker WI-7936 maps very closely to D2S177. thissequence tagged site (STS) corresponds to the gene for human cytochromeP4501B1 (CYP1B1) (Sutter, T. R. et al., J. Biol. Chem. 269:13092(1994)). A fifth gene encoding 9G8 splicing factor (SFRS7) wasidentified when a BLAST search determined that the expressed sequencetagged (EST) marker TIGR-A004S39 has been derived from the 3-primeuntranslated region of this gene (Popielarz, M. et al., J. Biol. Chem.270:17830 (1995)). This EST has already been mapped next to markerD2S177 on the chromosome 2 RH map (Hudson, T. et al., Science 270:1945(1995); Shuler, G. D. et al, Science 274:540 (1996)). All of these geneswere considered as potential candidate genes; coding sequences ofseveral of the genes were screened for mutations by a direct sequencingmethod.

Monolayers of human skin fibroblasts were maintained at 37° C. in a CO₂incubator, in media MEM (Gibco/BRL, catalog number 11095-080)supplemented with 10% Fetal Bovine Serum and antibiotics (penicillin G,streptomycin sulfate; Gibco/BRL catalog number 15140-015). Total RNA wasprepared with TRIzol reagent (Gibco/BRL) according to manufacturer'sprotocols. First strand synthesis was primed from 10 μl of the RNAsample with 50 ng random hexamers. Reaction was carried out in 20 mMTris-HCl (pH 8.4), 40 mM KCl, 2.5 mM MgCl₂, 0.5 mM from each dNTP, 0.01M DDT, and 200 U SuperScript II RT (Gibco) in a total volume of 20 μlfor 1 hour at 42° C. The coding sequence of the CYP1B1 gene wasamplified with the cDNA-based primer sets:

CYP1 (CYP1F 5'-GGTTCCTGTTGACGTCTTG-3' (SEQ ID NO. 2), CYP1R5'-CTTCCAGTGCTCCGAGTAG-3' (SEQ ID NO. 3));

CYP2 (CYP2F 5'-GTGGTGCTGAATGGCGAG-3' (SEQ ID NO. 4), CYP2R5'-TACTGCAGCCAGGGCATC-3' (SEQ ID NO. 5));

CYP3 (CYP3F 5'-GTGGCCAACGTCATGAGTG-3' (SEQ ID NO. 6), CYP3R5'-TCATAAAGGAAGGCCAGGAC-3' (SEQ ID NO. 7);

and CYP4 (CYP4F 5'-AGACTCGAGTGCAGGCAG-3' (SEQ ID NO. 8), CYP4R5'-TCCTCATCTCCGAAGATGGT-3' (SEQ ID NO. 9)). PCR amplification wascarried out with recombinant Taq polymerase (Gibco/BRL) according to themanufacturer's protocol. The amplified PCR fragments were purifieddirectly or from agarose gels with Wizard PCR preps DNA purificationsystem (Promega). Dye terminator sequencing with Taq Polymerase FS wasperformed on an ABI-373 sequencer (Perkin Elmer).

Initially, the coding sequences of the hSOS1 and PRKR genes werescreened; when no sequence variants were observed, mutation screening ofthe CYP1B1 gene was performed.

As a result of this screening, a 13 bp homozygous deletion (family 26;affected individual 10) that removed nucleotides 1410 to 1422 (i.e.,GAGTGCAGGCAGA (SEQ ID NO. 1)) from the coding sequences (i.e., exon III)of the CYP1B1 gene was identified. This mutation resulted in aframeshift that truncated the open reading frame by creating a prematurestop codon (TGA), 203 bp downstream of this deletion (or 68 amino acidsafter the last original amino acid Thr-354). In order to develop anassay for genomic DNA screening, the intron/exon junctions of this genewere determined. In order to recover the genomic region containing theCYP1B1 coding sequence, the cDNA-based primer sets CYP1-4 were used forlong-range PCR amplification. Total yeast DNA prepared from straincontaining YAC 806-F-8 (Hudson, T. et al., Science 270:1945 (1995))served as a template. Approximately 50 ng total yeast DNA were subjectedto PCR amplification with 20 pmol of each primer in a total volume of 50μl that consisted of 60 mM Tris-SO₄ (pH 9.1 at 25° C.), 18 mM (NH₄)₂SO₄, 1.5 mM MgSO₄, 0.2 mM each dNTP, and 2 μl eLONGase enzyme mix(Gibco/BRL). Amplification conditions consisted of 1 min initialdenaturation at 94° C., followed by 35 cycles of denaturation at 94° C.for 30 seconds, annealing at 53° C. for 30 seconds, and 6 minuteextension at 68° C. As a result, four PCR fragments ranging in size from1 kb to 3 kb were amplified. Purification and sequencing of thesefragments were performed as described above. Intron/exon junctions inthe CYP1B1 gene were identified by comparing the sequences of theamplified fragments to the reference cDNA sequence (Sutter, T. R. etal., J. Biol. Chem. 269:13092 (1994)). Three primer sets were assembledfor amplification of the CYP1B1 coding sequence from genomic DNA. PrimerCYP1F was paired with the intronic primer, 5'-CCTCCCAGAGGCTTTACCT-3'(SEQ ID NO. 10), for amplification of exon II under the conditionsdescribed for the long distance PCR (1.6 kb fragment). For amplificationof the coding region located in exon III, intronic primer5'-TAAGAATTTTGCTCACTTGC-3' (SEQ ID NO. 11) was paired with primer CYP4R(693 bp fragment). A 134 bp fragment containing the 3'-end of the CYP1B1coding sequence was amplified with primers 5'-TCAATGTCACTCTCAGAGAG-3'(SEQ ID NO. 12) and CYP4R. It was concluded that the CYP1B1 genecontains three exons and two introns, as shown in Table 1.

                  TABLE 1    ______________________________________    Exons in CYP1B1 Gene    Exon  Exon    Intron              3' Splice    No.   Size    Location  5' Splice Donor                                      Acceptor    ______________________________________    I      345    345/346   CGCAGgtcagt                                      cccagCATGG                            (SEQ ID NO. 13)                                      (SEQ ID NO. 14)    II    1044    1389/1390 ACCAGgtaaag                                      aacagGTATC                            (SEQ ID NO. 15)                                      (SEQ ID NO. 16)    III   3703*    ______________________________________     *3121 bp 3untranslated sequence

The genomic structure of the gene is shown in FIG. 2. The numerationreflects the cDNA sequence of the gene, and the coding regions are shownin black. The entire coding sequence of the gene is contained in exonstwo and three. The genomic structure of CYP1B1 determined here is inagreement with the result published recently (Tang, Y. M. et al., J.Biol. Chem. 271:28324 (1996)).

The presence of the 13 bp exonic deletion in family 26 and itscosegregation with the disease phenotype was confirmed by acrylamide gelelectrophoresis of a 124 bp PCR fragment that harbored the deletedregion. For rapid mutation screening, a 124 bp fragment containing the13 bp deletion was amplified from genomic DNA with primers:5'-CAAACAGGTATCCTGATGTG-3' (SEQ ID NO. 17) and CYP3R. The PCR productswere analyzed on polyacrylamide minigels consisting of 5% Acrylamide/Bissolution (19:1), 15% urea, and 1× TBE (see FIG. 3A). The same 13 bpdeletion was also detected and subsequently confirmed to segregate withthe disease phenotype in another family (family 17; FIG. 3A). The thirdband (*) observed in the heterozygote individuals represents aheteroduplex. Family 17 is a consanguineous marriage between an affectedfather and a normal mother. In this pedigree, it was determined that thefather is homozygote for the 13 bp deletion while the mother isheterozygote for the same deletion. Therefore, all the affected andnormal offspring have inherited a single copy of this deletion from hisfather alone, while the affected offspring, in addition, inherited a 13bp deletion from his mother.

A second mutation was observed by rapid mutation screening as describedabove, in another two families (families 10 and 11) who exhibited ahomozygous insertion of an extra cytosine base in a stretch of sixcytosines located between nucleotide positions of 1209 to 1214 in exonII (FIGS. 2 and 3B). This also proved to be a frameshift mutation thatcreated a premature stop codon (TGA), 106 bp downstream from the site ofthis insertion (or 36 amino acids downstream from the original aminoacid Pro-289).

Furthermore, a third mutation was detected in another consanguineousfamily (family 15). This is a much larger deletion that starts in intronII and removes a certain portion of coding sequences of exon III thatextends beyond the above-mentioned 13 bp deletion (FIGS. 2, 3C). Anassay for rapid mutation screening as described above was used. Themutation detected in exon II was amplified from genomic DNA withprimers: 5'-GACAAGTTCTTGAGGCACTGC-3' (SEQ ID NO. 18) and5'-ACGTTCTCCAAATCCAGCC-3' (SEQ ID NO. 19) The amplified fragments wereelectrophoresed on sequencing type acrylamide gel under denaturingconditions (1 M urea, 50-54° C.). Gels were visualized by silverstaining. From the PCR amplification pattern, it is shown that the5-prime end of exon III and the adjacent intronic region are deleted,but that the 3-prime end of exon III has remained intact (FIG. 3C). Thetop band represents a 1.6 kb fragment containing the entire exon II andthe adjacent 5-prime intron. The second fragment contains the entirecoding sequence located in exon III. The smallest amplification productcontains 134 bp from the 3'-end of the CYP1B1 coding sequence. As the3-prime splice acceptor site of intron II has been deleted, thismutation is expected to interfere with the normal splicing of the CYP1B1gene, resulting in synthesis of either truncated protein, or nullallele.

A fourth mutation was detected in an individual. This mutation is a 10base pair duplication of nucleotides 1546-1555 (TCATGCCACC, SEQ ID NO.20), which results in a frame shift mutation that created a prematurestop codon. The premature stop codon resulted in a deletion of all aminoacits after amino acid 403 (deletion of the last 140 amino acids).

Analysis of 470 chromosomes from randomly selected normal individuals(330 Turkish and 140 other Caucasians) failed to detect the presence ofthe four mutant alleles described above, making it less likely thatthese sequence variants represent rare polymorphism. These mutationswere only observed in 18 affected subjects but not the normal members ofa total of 7 families (including 5 consanguineous families), and, thenormal population from which these families are ascertained did notcarry these mutations, strongly suggesting that the CYP1B1 gene is thegene for the GLC3A locus on 2p21.

A fifth mutation was a single base deletion of cytosine at nucleotide1737. This mutation similarly resulted in a frame shift mutation thatcreated a premature stop codon. The premature stop codon resulted in thedeletion of 80 amino acids (all amino acids after amino acid 463).

A sixth mutation was a single base, G→T transition of nucleotide 1188.This mutation created a premature TAA stop codon after amino acid 281,removing 263 amino acids from the full-length protein.

A single base pair C→T transition at nucleotide 1482 was also detected.This change results in a change of the encoded amino acid from prolineto leucine.

Ten additional mutations have also been identified. These mutations wereeither sporadic or familial, and found in one or more individuals orfamilies from varying geographical populations (families of Turkish, US,Pakistani, UK, Hispanic or French Canadian origin). The followingmutations were found: a single base change (a G→C transition) atnucleotide 517, resulting in a change of the encoded amino acid fromtryptophan to cysteine; a single base change (a G→A transition) atnucleotide 528, resulting in a change of the encoded amino acid fromglycine to glutamic acid; an insertion of a thymine base at nucleotide846, causing a frame shift mutation that resulted in the deletion of 377amino acids from the carboxy terminus of the protein; a single basechange (a G→T transition) at nucleotide 1439, resulting in a change ofthe encoded amino acid from glycine to tryptophan; a single base change(a G→A transition) at nucleotide 1505, resulting in a change of theencoded amino acid from glutamic acid to lysine; a single base change (aG→A transition) at nucleotide 1515, resulting in a change of the encodedamino acid from arginine to histidine; a single base change (a C→Ttransition) at nucleotide 1656, resulting in a change of the encodedamino acid from proline to leucine; a deletion of a single base (G) atnucleotide 1691, causing a frame shift mutation that resulted in thedeletion of 95 amino acids from the carboxy terminus of the protein; asingle base change (a C→T transition) at nucleotide 1751, resulting in achange of the encoded amino acid from arginine to tryptophan; or aduplication of 27 nucleotides, beginning at nucleotide 1749, causing aframe shift mutation that resulted in deletion of 76 amino acids fromthe carboxy terminus of the protein.

The mutations described above are summarized in Table 2. The "locationin protein" in Table 2 indicates the region of the protein's structurehaving the mutation; and the "restriction sites" indicates the additionor deletion of restriction sites. The changes in the restriction sitescan be used, for example, to perform mutation analysis by restrictiondigestion, as described above. The first five mutations are within exonII; the remaining mutations affect exon III.

                                      TABLE 2    __________________________________________________________________________    Mutations Associated with Primary Congenital Glaucoma                        Location in                               COOH-- Amino                                       Restriction    Position**          DNA change                Effect of change                        protein                               acids deleted                                       sites    __________________________________________________________________________    517   G-->C Trp(57)-->Cys                        hinge region                               n/a     n/a    528   G-->A Gly(61)-->Glu                        hinge region                               n/a     + TaqI    846   insert T                frameshift                        n/a    377     - XhoI    1187  G-->T Glu(281)-->stop                        n/a    262     n/a    1209  insert C                frameshift                        n/a    254     n/a    exon  large splicing error                        n/a    n/a     n/a    II, III          deletion    1410  delete 13                frameshift                        n/a    189     - XhoI          nucleotides    1439  G-->T Gly(365)-->Trp                        J-helix                               n/a     n/a    1482  C-->T Pro(379)-->Leu                        K-helix                               n/a     n/a    1505  G-->A Glu(387)-->Lys                        K-helix                               n/a     n/a    1515  G-->A Arg(390)-->His                        K-helix                               n/a     - CfoI    1546  duplicate 10                frameshift                        n/a    140     + NIa III          aa    1656  C-->T Pro(437)-->Leu                        Meander                               n/a     n/a    1691  G deletion                frameshitt                        n/a    95      n/a    1751  C-->T Arg(469)-->Trp                        Heme-binding                               n/a     - AciI    1749  duplicate 27                frameshift                        n/a    76      + BlaI          aa    1737  C deletion                stop codon                        n/a    80      n/a    __________________________________________________________________________     **The position is the nucleotide that is altered, inserted or deleted, or     the nucleotide at which a duplication begins, unless otherwise indicated.

If a stable protein product is produced from the mutated genes describedabove, the products are expected to lack from 80 to 377 amino acids fromthe --COOH terminus. This segment harbors the invariant cysteine of allknown cytochrome P450 amino acid sequences (i.e., Cys-470 of CYP1B1).This residue provides the axial heme ligand that defines many of thefunctional and spectral characteristics of the cytochrome P450 proteins(Hudson, T. et al., Science 270:1945 (1995); Gonzalez, F. J., Pharmacol.Rev. 40:243 (1989))). The adjacent residues Phe-463 to Gly-472,correspond to the protein sequence pattern that identifies the cysteineheme-iron ligand signature sequence of cytochrome P450 (PROSITEaccession PS00086; (Hudson, T. et al., Science 270:1945 (1995); Bairoch,A., Nucl. Acids Res. 20 (suppl) :2013 (1992)). The removal of thisessential region is expected to interfere with the ability of thetruncated molecules to perform normal physiologic functions.

A G to C transversion at nucleotide 1640 of the CYP1B1 coding sequence,which changes Val-432 to Leu, has also been detected (FIG. 2). Thischange was found to create and Eco57I restriction site, thus providing arapid screening method. A total of 70 normal individuals (47 Turkish and23 other Caucasians) were screened for the presence or absence of thischange. Thirty-six individuals (51.4%) were found to be heterozygote forthis change. Of the remaining 34 homozygote individuals (48.6%), 27subjects had leucine and 7 had valine. The amino acid position wherethis change has occurred is not part of the CYP1B1 conserved sequence;further, both valine and leucine are neutral and hydrophobic amino acidswith similar aliphatic side groups differing only by a single CH₂ group.Thus, this change represents a polymorphism that is unrelated to theprimary congenital glaucoma phenotype.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described specifically herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 20    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 13 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    #      13    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    # 19               TTG    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    # 19               TAG    - 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    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    # 20               TTGC    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    # 20               AGAG    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 11 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    #       11    - (2) INFORMATION FOR SEQ ID NO:14:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 10 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    #        10    - (2) INFORMATION FOR SEQ ID NO:15:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 11 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    #       11    - (2) INFORMATION FOR SEQ ID NO:16:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 10 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    #        10    - (2) INFORMATION FOR SEQ ID NO:17:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    # 20               TGTG    - (2) INFORMATION FOR SEQ ID NO:18:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    #21                ACTG C    - (2) INFORMATION FOR SEQ ID NO:19:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    # 19               GCC    - (2) INFORMATION FOR SEQ ID NO:20:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 10 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    #        10    __________________________________________________________________________

What is claimed is:
 1. A method of diagnosing primary congenitalglaucoma in an individual, comprising detecting a deletion of nucleotide1691 in the human cytochrome P4501B1 gene, wherein the presence of thedeletion is indicative of primary congenital glaucoma.
 2. A method ofdiagnosing primary congenital glaucoma in an individual, comprisingdetecting an insertion of a single thymine residue at nucleotide 846 inthe human cytochrome P4501B1 gene, wherein the presence of the insertionis indicative of primary congenital glaucoma.
 3. A method of diagnosingprimary congenital glaucoma in an individual, comprising detecting aduplication of nucleotides 1749-1775 in the human cytochrome P4501B1gene, wherein the presence of the duplication is indicative of primarycongenital glaucoma.
 4. A method of diagnosing primary congenitalglaucoma in an individual, comprising detecting a change of nucleotide517 from G to C in the human cytochrome P4501B1 gene, wherein thepresence of the insertion is indicative of primary congenital glaucoma.5. A method of diagnosing primary congenital glaucoma in an individual,comprising detecting a change of nucleotide 528 from G to A in the humancytochrome P4501B1 gene, wherein the presence of the insertion isindicative of primary congenital glaucoma.
 6. A method of diagnosingprimary congenital glaucoma in an individual, comprising detecting achange of nucleotidc 1439 from G to T in the human cytochromc P4501B1gene, wherein the presence of the insertion is indicative of primarycongenital glaucoma.
 7. A method of diagnosing primary congenitalglaucoma in an individual, comprising detecting a change of nucleotide1505 from G to A in the human cytochrome P4501B1 gene, wherein thepresence of the insertion is indicative of primary congenital glaucoma.8. A method of diagnosing primary congcnital glaucoma in an individual,comprising detecting a change of nucleotide 1515 from G to A in thehuman cytochrome P4501B1 gene, wherein the presence of the insertion isindicative of primary congenital glaucoma.
 9. A method of diagnosingprimary congenital glaucoma in an individual, comprising detecting achange of nucleotide 1656 from C to T in the human cytoclromc P4501B1gene, wherein the presence of the insertion is indicative of primarycongenital glaucoma.
 10. A method of diagnosing primary congenitalglaucoma in an individual, comprising detecting a change of nucleotide1751 from C to T in the human cytochrome P4501B1 gene, wherein thepresence of the insertion is indicative of primary congenital glaucoma.